A bacterial type I polyketide synthase (PKS) is comprised of an intriguing set of complex multifunctional proteins that along with allied enzymes generate structurally complex and clinically important natural products via a modular multi-step process. Numerous systems of this type have been discovered over the past decade, paving the way to engineered PKSs that generate novel natural products. Access to affordable high throughput genome sequencing of diverse microbial systems is revealing new PKS, non- ribosomal peptide synthetase (NRPS) and mixed PKS-NRPS systems at an ever-increasing rate. Moreover, bioinformatic tools to predict the structural outcome of these metabolic systems are providing rapid access to new natural products. Despite increasing access to new information, obtaining a detailed biochemical understanding of PKS-NRPS systems is necessary to test functional predictions and demands the application of rigorous experimental approaches. Understanding these details will not only expand our basic knowledge of PKS-NRPS molecular machines, but also provide new strategies to manipulate them to expand chemical diversity. Such systems are attractive due to their potential to create new chemotypes with valuable applications in drug discovery and development. Despite remarkable progress, an understanding of the molecular mechanisms, catalytic activities, kinetic properties, substrate specificity and protein-protein recognition in both natural and hybrid PKSs remains limited. This competing renewal application proposes to employ the versatile and well-characterized Streptomyces venezuelae pikromycin PKS, as well as a series of additional pathways whose detailed analysis has been initiated during the previous cycle of support. These systems each bear fascinating biochemical attributes that will expand our understanding of the specificity and structural features that lead to functional activity within and between native and hybrid PKS modules. Our objectives and approach will focus on assessing the molecular details of polyketide chain initiation, elongation, 2-branching and termination that lead to the remarkable chemical diversity of polyketide natural products. This detailed biochemical analysis, and the integration of structural biology to probe substrate specificity and synthetic chemistry to develop chemoenzymatic approaches will allow pursuit of our long term objective of engineering PKS systems that efficiently generate novel structures with significant potential as therapeutic agents. Specific aims include: I. Molecular Analysis of Modular Polyketide Synthases. Design and employ synthetic substrates and Pik, DEBS, and Tyl terminal modules to explore selectivity and tolerance in chain loading, elongation and processing. II. Molecular recognition as the basis for protein-protein interactions in modular PKSs. Explore molecular parameters of docking selectivity by designing and constructing effective pathways using native, and heterologous docking domain combinations. III. Analysis of the molecular basis for termination in modular systems. Explore the determinants of macrolactone formation vs. hydrolysis by the terminating thioesterases in the PKSs for pikromycin, erythromycin, tylosin, tautomycetin, curacin, and carmabin. IV. Analysis of new catalytic domains and molecular interactions in modular PKSs that synthesize 2-branched products. Pursue analysis of the bryostatin biosynthetic system (Bry) including HMG synthase and 2-branching leading to the modified pyrone ring system. Explore the basis for acyl-ACP cognate enzyme interactions in Bry including ACPD::HMGS, ACPD::KS, and KS::HMGS).